A protocol to construct RNA-protein devices for photochemical translational regulation of synthetic mRNAs in mammalian cells

Summary Here, we describe a protocol for the translational regulation of transfected messenger RNAs (mRNAs) using light in mammalian cells. We detail the steps for photocaged ligand synthesis, template DNA preparation, and mRNA synthesis. We describe steps for mRNA transfection, treatment of cells with a photocaged ligand followed by light irradiation, and analysis of the transgene expression. The protocol enables spatiotemporally regulated transgene expression without the risk of insertional mutagenesis. For complete details on the use and execution of this protocol, please refer to Nakanishi et al. (2021).

Here, we describe a protocol for the translational regulation of transfected messenger RNAs (mRNAs) using light in mammalian cells. We detail the steps for photocaged ligand synthesis, template DNA preparation, and mRNA synthesis. We describe steps for mRNA transfection, treatment of cells with a photocaged ligand followed by light irradiation, and analysis of the transgene expression. The protocol enables spatiotemporally regulated transgene expression without the risk of insertional mutagenesis.
Publisher's note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

SUMMARY
Here, we describe a protocol for the translational regulation of transfected messenger RNAs (mRNAs) using light in mammalian cells. We detail the steps for photocaged ligand synthesis, template DNA preparation, and mRNA synthesis. We describe steps for mRNA transfection, treatment of cells with a photocaged ligand followed by light irradiation, and analysis of the transgene expression. The protocol enables spatiotemporally regulated transgene expression without the risk of insertional mutagenesis. For complete details on the use and execution of this protocol, please refer to Nakanishi et al. (2021).

BEFORE YOU BEGIN
Selection of reporter genes to be regulated Timing: 1 h 1. If you will use a flow cytometer to analyze the translational activation or repression of the target mRNAs, check the laser-filter sets of the flow cytometer and select fluorescent proteins with excitation and emission wavelength peaks that are close to those available on the flow cytometer. Not only excitation and emission wavelength but also other properties such as brightness and cytotoxicity should also be considered. This may be eased by using a fluorescent protein database, such as FPbase (Lambert, 2019). 2. Alternatively, if you will use a luminometer, you can use luciferase genes as reporters.
Note: It depends on the type of Caliciviral VPg-based Translational activator (CaVT) whether 1xMS2(U)site1 or site2 mRNA is preferable for the translational activation. We previously showed that 1xMS2(U)site2 mRNA is preferable for non-split type CaVT-mediated translational activation. On the other hand, when split CaVT is used, 1xMS2(U)site1 mRNA is preferable (Nakanishi and Saito, 2020). However, both types of mRNAs can be translationally activated by both types of CaVT.
Note: We adopt the procedure of adding part of the UTRs in the 1 st round PCR and the rest of the UTRs and T7 promoter in the 2 nd round PCR because long primers are expensive or cannot be ordered. However, if ordering long primers is not a problem, it is also possible to prepare template DNAs directly from pDNAs by a single PCR. Timing: 10 h synthesis of TMP-HL (1) CRITICAL: The synthesis scales do not always match between steps, but these scales have been optimized and changing them may result in reduced yields.

KEY RESOURCES
The following steps describe the synthesis and characterization of TMP-HL (1), see Figure 2.
Note: All the procedures should be conducted in a fume food. Unless noted all rotary evaporation steps are carried out at 25 C.

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Pause point: At this point, the product can be stored at -20 C for at least 2 years.
2. Synthesis of TMP-HL (1). a. Weigh 49 mg (0.104 mmol) of compound 4 in a 50 mL two-neck round-bottom flask containing a magnetic stirring bar. b. Add 1 mL of CH 2 Cl 2 and 0.5 mL of trifluoroacetic acid (TFA). c. Stir the reaction mixture at room temperature for 1 h. d. Add 1 mL of toluene to the mixture and remove the solvent using a rotary evaporator under reduced pressure at 40 C. Repeat this process two more times to afford a deprotected form of compound 4 as a colorless oil. e. Equip the flask with an argon-filled balloon. f. Dissolve the crude product with 2 mL of anhydrous DMF. g. Add 31.2 mg (0.083 mmol) of 5-(4-((2,4-diaminopyrimidin-5-yl)methyl)-2,6-dimethoxyphenoxy)pentanoic acid ( Pause point: The product can be stored at -20 C for at least 2 years.

Synthesis of photocaged TMP-HL (2)
Timing: 19 h for synthesis of compound 7 Timing: 10 h for synthesis of compound 8 Timing: 18 h for synthesis of photocaged TMP-HL (2) The following steps describe the synthesis and characterization of photocaged TMP-HL (2), see Figure 3.
Note: All the procedures should be operated in a fume food.  d. Slowly add 68 mg (0.25 mmol) of 4,5-dimethoxy-2-nitrobenzyl chloroformate at 0 C to the flask. e. Stir the reaction mixture at room temperature for 9 h under argon. f. Dilute the reaction mixture with 20 mL of CH 2 Cl 2 and wash the organic layer with water (20 mL 3 2) and brine (20 mL 3 1) in a separatory funnel. Collect the organic layer and dry it with anhydrous Na 2 SO 4 . After filtration to remove Na 2 SO 4 , condense the organic layer until the solvent is no longer detectable using a rotary evaporator under reduced pressure. g. Purify the crude product using silica gel column chromatography (100 mL bed volume, washed with 200 mL CHCl 3 , 600 mL 20:1 CHCl 3 /MeOH, then eluted with 800 mL 50:1 CHCl 3 /MeOH)). h. Collect the fractions and remove solvent using rotary evaporator at 30 C. i. Dry the sample under reduced pressure at RT for at least 1 h to afford compound 7 (18 mg, yield 11%) as a pale yellow solid. j. Characterize the product by 1 H NMR spectroscopy. 1    Pause point: At this point, the product can be stored at -20 C for at least 2 years.
5. Synthesis of photocaged TMP-HL (2). a. Weigh 12.6 mg (27 mmol) of compound 4 in a 50 mL two-neck round-bottom flask containing a magnetic stirring bar. b. Add 2 mL of CH 2 Cl 2 and 2 mL of TFA. c. Stir the reaction mixture at room temperature for 1 h. d. Add 1 mL of toluene to the mixture and remove the solvent using a rotary evaporator under reduced pressure. Repeat this process two more times to afford a deprotected form of compound 4 as a colorless oil. e. Equip the flask with an argon-filled balloon. f. Dissolve the crude product with 1 mL of dry DMF. g. Add 11 mg (18 mmol aqueous HCl solution (20 mL 3 1) and saturated NaHCO 3 solution (20 mL 3 1) in a separatory funnel. Collect the organic layer and dry it with anhydrous Na 2 SO 4 . After filtration to remove Na 2 SO 4 , condense the organic layer using a rotary evaporator under reduced pressure at 35 C. k. Purify the crude product by reversed-phase HPLC using a semi-preparative C18 column (a linear gradient of MeCN containing 0.1% TFA and 0.1% aqueous TFA solution) to afford photocaged TMP-HL (2) (9.5 mg, yield 55%) as a white solid after lyophilization. l. Characterize the product by 1 H NMR spectroscopy and ESI-MS. 1  Pause point: The product can be stored at -20 C for at least 2 years.

Synthesis of photocaged TMP (3)
Timing: 14 h The following steps describe the synthesis and characterization of photocaged TMP (3), see Figure 4.
Note: All the procedures should be operated in a fume food.   Pause point: The product can be stored at -20 C for at least 2 years.

Timing: 3-4 h
Template DNAs for IVT are prepared by two rounds of PCR ( Figure 1). Two types of DNA fragments are obtained in the 1 st round. One is the DNA containing the translational regulation-target or CaVT gene (hereafter called open reading frames (ORF)) flanked with the partial 5 0 and 3 0 UTR sequences. The other is the DNA containing the 3 0 UTR sequence. In the 2 nd round of PCR, these two DNA fragments are fused and the sequences of T7 promoter, 5 0 UTR, and poly(A) tail are added. Although the protocol uses PrimeSTAR Max DNA polymerase, this can be substituted with another high-fidelity PCR enzyme following the manufacturer's recommended protocol.
7. 1 st round PCR. a. To amplify the ORF flanked with the partial 5 0 and 3 0 UTR sequences, prepare the PCR mixtures shown below.

Reagent Final concentration Amount
PrimeSTAR Max Premix (23 In the case of amplifying the ORF-containing DNA, a 20-cycles reaction is usually enough, as the 2 nd round PCR needs only a small amount of the DNA. 8. After the PCR, add 1 mL of DpnI to the PCR mixtures and incubate them at 37 C for 0.5-1.0 h to remove template pDNAs. In the case of a PCR to amplify 3 0 UTR, this step can be ignored. 9. Mix a portion of the PCR mixtures (e.g., 2 mL) with loading dye and perform the electrophoresis using 1.2% agarose gels (100 V 25 min). Then, stain the gels with a gel-staining reagent (e.g., Midori Green Advance) and capture images of gels to confirm the size of the amplified DNAs. b. Incubate the IVT reaction mixture at 37 C for 4-6 h.
Note: We recommend using a constant-temperature incubator rather than a block heater for the IVT reaction. Incubation with a block heater for 4-6 h may cause water evaporation followed by condensation on the tube lid, which alters the concentration of the IVT reaction components.
15. Remove the template DNA by adding 1 mL of TURBO DNase (a component of MEGAscript T7 Transcription Kit) to each IVT reaction mixture and incubating at 37 C for 30 min. 16. Purify each mRNA using an RNA purification kit of your choice according to the manufacturer's instructions (e.g., NEB Monarch RNA Cleanup Kit). 17. Dephosphorylate the mRNA using alkaline phosphatase (rApid alkaline phosphatase is given as an example) by mixing the components of the dephosphorylation reaction as shown below. Then, incubate the reaction mixture at 37 C for 30 min.
18. Purify the mRNAs using an RNA purification kit according to the manufacturer's instructions. 19. Measure the concentration of the purified mRNAs by absorbance spectroscopy. 20. Check the size and the quality of the purified mRNAs using Bioanalyzer and RNA 6000 pico kit according to the manufacturer's instructions. Alternatively, other methods (e.g., Denaturing PAGE or Microchip Electrophoresis) could be used to analyze the sample purity and size.
Note: 2xScMS2(C) mRNA has a highly stable secondary structure, which is hard to denature, and can show two peaks. For the other mRNAs, only a single peak should be observed (Figure 5). See troubleshooting problem 2 if multiple peaks are observed.

mRNA transfection, light irradiation, and expression analysis
Timing: 3 days 21. Seed the appropriate number of cells (e.g., 5 3 10 4 HeLa cells in 500 mL/well of DMEM containing FBS and antibiotics) onto 24-well clear flat-bottom plates. Usually, 70%-90% confluent at transfection is suitable. To compare irradiated and non-irradiated conditions, at least two plates are needed. After seeding, incubate the cells at 37 C in a 5% CO 2 incubator. Most mRNAs should show a single peak, but 2xScMS2(C) mRNAs tend to show two peaks.

Reagent Amount
Opti-MEM 25 mL/well Lipofectamine MessengerMAX 1 mL/well c. Incubate for 10 min at room temperature. Prepare the transfection complex by mixing the diluted mRNA mixture and the diluted transfection reagent together and incubate for 5 min at room temperature. d. Add the transfection complex directly to the medium above the plated cells. e. Incubate the cells at 37 C in a 5% CO 2 incubator for 3 h. 23. Prepare medium containing 250 nM photocaged TMP-HL (for split CaVT) or 10 mM photocaged TMP (for DD-CaVT). Medium containing TMP-HL or TMP without photocage can be used as a positive control. Avoid light irradiation to the photocaged ligands. 24. Three hours after the transfection, change the medium to the photocaged ligand-containing one. To avoid decaging of the ligands in the unirradiated control plate, shield the plate from light (e.g., by wrapping the plate with aluminum foil). 25. Place the cell culture plates directly onto an HP-30LM and irradiate with UV light (wavelength: 365 nm) from the bottom of the plates for 3-7 min. To avoid UV exposure to the experimenters, we recommend doing this procedure in a clean bench equipped with a UV shield. If a UV lamp other than HP-30LM is used for UV light irradiation, the irradiation time should be optimized depending on the light intensity. In the case of HP-30LM, the light intensity measured at the bottom of the plate by a photodiode power sensor was approximately 3.34 mW/cm 2 . 26. Incubate the cells at 37 C in a 5% CO 2 incubator for 1 day. 27. Analyze the gene expression by a method suitable for the gene that is encoded by the transfected mRNA. An example of the procedure to analyze fluorescent protein expression using a flow cytometer is shown below. a. Detach the cells using 200 mL/well of 0.25% Trypsin/EDTA or other appropriate methods. Then, suspend the detached cells by adding 500 mL/well of the medium. b. Strain the cells using a cell strainer. Because the cells can aggregate over time, we recommend straining the cells immediately before measuring the fluorescence by flow cytometry. c. Measure the fluorescence by flow cytometry according to the manufacturer's instruction.
Note: Conditions to be tested are listed below. Optional: Analyze the data of the flow cytometry using appropriate software (e.g., FlowJo).

EXPECTED OUTCOMES
In the case of translational activation by split CaVT or DD-CaVT, cells treated with a photocaged ligand should show a light-dependent increase in the production of protein from 1xMS2(U)site1 or site2 mRNA (Nakanishi et al., 2021) (Figures 6 and 7).
Conversely, in the case of translational repression by DD-CaVT, cells treated with a photocaged ligand should show a light-dependent decrease in the production of protein from 2xScMS2(C) mRNA ( Figure 8).

LIMITATIONS
Even in the translation-OFF state, there is usually leaky translation. Such leaky expression may affect cells even in the translation-OFF state should you want to regulate the mRNA encoding the protein with physiological activity. In addition, the expected fold-change by light irradiation is approximately three, which may be insufficient for some applications.

Potential solution
Optimize the PCR conditions (e.g., annealing temperature, PCR enzyme, and ramp rate) or purify the main product using a DNA gel extraction and purification kit.

Problem 2
Two or more peaks (in the case of 2xScMS2(C) mRNA, three or more peaks) are observed in the mRNA quality check by Bioanalyzer.

Potential solution
Verify the absence of PCR by-products or residual pDNAs in the IVT template DNAs by running a larger amount of IVT template DNAs in the agarose gel electrophoresis. If PCR by-products are

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observed, optimize the PCR conditions, as described in troubleshooting problem 1. If residual pDNAs are observed, increase the reaction time or the enzyme concentration of the DpnI digestion.
If only a single band is observed, check the RNA secondary structures by a secondary structure prediction tool, such as ParasoR (Kawaguchi and Kiryu, 2016) or MXfold2 (Sato et al., 2021). Stable stemloop structures in mRNAs may cause multiple peaks even when the solution contains a single type of mRNA.

Problem 3
Transfection efficiency is too low.

Potential solution
Change the transfection condition. For example, extending the duration from transfection to medium change, using a transfection reagent other than Lipofectamine MessengerMAX (e.g., Stem-Fect RNA Transfection Kit (ReproCELL) or TransIT-mRNA Transfection Reagent (Takara Bio)), or using an electroporator instead of a transfection reagent.

Problem 4
The light-unirradiated group shows a similar translation level to the light-irradiated group and the positive control (a ligand without photocage-added) group. Only the negative control (no ligand addition) group shows a low (in the case of translation activation) or high (in the case of translational repression) level.

Potential solution
The photocaged ligand may be uncaged due to light exposure during storage or there may be a failure in the caging reaction. Confirm the photocaged ligand by mass spectrometry. If the ligand is already uncaged, prepare a new lot of the photocaged ligand. To avoid the uncaging of the photocaged ligand, dispense and store it in a light-shielded condition.

Problem 5
The light-irradiated group shows a similar translation level to the light-unirradiated group and the negative control (no ligand addition) group. Only the positive control (a ligand without photocage-added) group shows a high (in the case of translation activation) or low (in the case of translational repression) level.

Potential solution
Increase the duration of the light irradiation.

Problem 6
The positive control (a ligand without photocage-added) group shows a similar translation level to the negative control (no ligand addition) group.

Potential solution
Co-transfect the target mRNA and the conventional (unsplit and no DD-fused) CaVT mRNA. If the conventional CaVT can translationally activate or repress the target mRNA translation, verify the quality and the preparation procedure of split CaVT or DD-CaVT. Even if the conventional CaVT cannot alter the target mRNA translation, verify the quality and the preparation procedure of the target mRNAs.  Figure 7, the photolysis of the photocaged TMP stabilizes DD-CaVT. Then, the stabilized DD-CaVT binds 2xScMS2(C) mRNA. Different from the case of 1xMS2(U)site2 mRNA, the binding between 2xScMS2(C) mRNA and DD-CaVT is very strong, which results in translational repression rather than activation. To keep the basal translation level of 2xScMS2(C) mRNA high, it is capped with ARCA, a translationally active cap analog.

Problem 7
The translation level can be regulated by light irradiation, but the absolute protein production is too low even in the translation-ON state.

Potential solution
Except for the case of 1xMS2(U)site1 and site2 mRNAs, the absolute protein production may be improved using CleanCap AG reagent instead of ARCA. Note that CleanCap AG reagent needs the modified T7 promoter sequence (TAATACGACTCACTATAAGG) in IVT template DNAs instead of the usual T7 promoter sequence (TAATACGACTCACTATAGGG).
Removal of double-stranded RNA by-products (Baiersdö rfer et al., 2019) and optimization of the transfection conditions and codon usage may also improve absolute protein production. If you are using a target mRNA encoding a fluorescent protein, a brighter protein is also an option.

RESOURCE AVAILABILITY
Lead contact Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead and Technical Contacts, Hirohide Saito (hirohide.saito@cira.kyoto-u.ac.jp) and Hideyuki Nakanishi (nakanishi.hideyuki.3m@kyoto-u.jp).
Materials availability pDNAs necessary for split CaVT and DD-CaVT mRNA preparation can be obtained from Addgene. Other materials are commercially available.

Data and code availability
This study did not generate any datasets or codes.